This invention relates to means of immobilizing nucleic acids such as DNA and RNA on solid supports. Immobilized nucleic acids find particular use as probes for determining the presence of particular polynucleotide sequences by hybridization with complementary single stranded polynucleotides. Nucleic acid hybridization is useful as an analytical method in the fields of human and veterinary medicine, agriculture, and food science, among others. In particular, the method can be used to detect and identify etiological agents such as bacteria and viruses, to screen microbes for antibiotic resistance, and to detect malignant cells.
Hybridization assays commonly involve the immobilization of either the nucleic acids present in the test sample or the probe nucleic acid. Such solid-phase techniques are concluded with the detection of hybrids formed on the immobilized phase between the probe and complementary sample polynucleotides. By far the most commonly used matrix for immobilization of nucleic acids in these methods has been microporous nitrocellulose membranes. More recently, microporous nylon membranes have become popular because they have better mechanical strength than nitrocellulose. Some manufacturers have introduced positive ionic groups such as quaternary ammonium ions into nylon membranes to improve their wetting proprieties. All of the known nitrocellulose and nylon membranes used to immobilize nucleic acids require high salt to adsorb the polynucleotides to their surface and baking at around 80.degree. C. to permanently fix the adsorbed DNA or RNA.
Detection of resulting immobilized hybrids formed on the solid matrix is conveniently accomplished by the addition of a detectable protein reagent that binds specifically to the hybrids. Normally such protein reagent will comprise an antibody or other binding protein that is specific for binding to a ligand moiety on the probe nucleic acid or to the unique configuration of the hybrid itself. Examples of the former are the detection of probe nucleic acids bearing a biotin or a hapten group by binding of avidin or anti-hapten antibody. Examples of the latter are the use of antibodies selective for DNA.RNA, or RNA.RNA duplexes or intercalated or otherwise antigenically modified duplexes. The specifically binding protein reagent is labeled with a detectable component, commonly an enzyme.
The problem with use of enzyme-labeled, or otherwise detectable, protein reagents to determine hybridization on the conventionally known solid matrices is nonspecific adsorption of such reagent. This nonspecific binding limits the sensitivity of the overall assay procedure. Accordingly, there is a need for better solid matrices for immobilizing nucleic acids, matrices which do not require high salt or baking in order to obtain efficient and stable immobilization. Further, such new matrices are needed particularly for use in hybridization assays, and particularly where resulting hybrids are detected with labeled protein reagents. Also, since hybridization procedures typically require several incubation and washing steps, the known microporous membranes are not amenable to rapid processing because they are fragile and difficult to handle. A solid, more rigid support material would overcome these problems.